Lehman E D, Joyce J G, Freymeyer D K, Bailey F J, Herber W K, Miller W J
Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, PA 19486.
Biotechnology (N Y). 1993 Feb;11(2):207-12. doi: 10.1038/nbt0293-207.
Tick anticoagulant peptide (TAP) is a 60 amino acid peptide (Mr = 6977, pI = 4.9) found in the saliva of the soft tick Ornithodorous moubata that specifically inhibits blood coagulation factor Xa (fXa). A recombinant form of TAP (rTAP) secreted by Saccharomyces cerevisiae was purified from 200 liters of fermentation broth with POROS, strong cation exchange (SCX) and reversed-phase high performance liquid perfusion chromatography (RP-HPLC) media (20 microns nominal particle diameter). The higher linear flow rates and dynamic capacities, as well as low back pressures, obtained with perfusion chromatography media permitted 37.5 g of rTAP to be efficiently captured and significantly enriched from 400 liters of diafiltered fermentation broth (24.3 g yield) with 1.25 liter of SCX media using a low pressure column and peristaltic pump in 4.5 hours. Subsequently, 16.7 g of rTAP obtained from the capture step was purified in a high-resolution mode with the same SCX media, after the media was cleaned and repacked into an 800 ml high-pressure column. By doing multiple rapid cycles at high linear flow rates, preparative-scale high-resolution purification of multi-gram amounts of the peptide was done on this relatively small column in only 10.5 hours. Finally, the peptide was desalted and decolorized on a 200 ml RP-HPLC column of perfusion chromatography media by doing multiple rapid cycles. After lyophilization, 12 g of peptide (46.9% yield) was obtained that was > 96% homogeneous by several analytical criteria and fully active in inhibiting blood coagulation factor Xa (fXa).(ABSTRACT TRUNCATED AT 250 WORDS)
蜱抗凝肽（TAP）是一种由60个氨基酸组成的肽（分子量为6977，等电点为4.9），存在于软蜱钝缘蜱的唾液中，可特异性抑制凝血因子Xa（fXa）。通过酿酒酵母分泌的重组形式的TAP（rTAP），使用POROS、强阳离子交换（SCX）和反相高效液相灌注色谱（RP-HPLC）介质（标称粒径20微米）从200升发酵液中纯化得到。灌注色谱介质具有较高的线性流速和动态容量，以及较低的背压，使用低压柱和蠕动泵，在4.5小时内，用1.25升SCX介质可从400升渗滤发酵液中高效捕获并显著富集37.5克rTAP（产率24.3克）。随后，在将介质清洗并重新装填到一个800毫升高压柱后，用相同的SCX介质以高分辨率模式纯化从捕获步骤中获得的16.7克rTAP。通过在高线性流速下进行多次快速循环，仅在10.5小时内就在这个相对较小的柱上完成了多克量肽的制备规模高分辨率纯化。最后，通过多次快速循环，在一个200毫升的RP-HPLC灌注色谱介质柱上对该肽进行脱盐和脱色。冻干后，获得了12克肽（产率46.9%），通过多种分析标准，其纯度>96%，并且在抑制凝血因子Xa（fXa）方面具有完全活性。（摘要截断于250字）
